scholarly journals High-performance GFP-based calcium indicators for imaging activity in neuronal populations and microcompartments

2018 ◽  
Author(s):  
Hod Dana ◽  
Yi Sun ◽  
Boaz Mohar ◽  
Brad Hulse ◽  
Jeremy P. Hasseman ◽  
...  
Keyword(s):  
Calcium Imaging ◽  
High Performance ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Neuronal Cell ◽  
Wide Field ◽  
Decay Kinetics ◽  
Calcium Indicators ◽  
Large Populations

AbstractCalcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics, and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and tracking large populations of neurons using 2-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

scholarly journals In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells and No Detectable Photobleaching

2021 ◽  
Author(s):  
Hannah S Wirtshafter ◽  
John F Disterhoft
Keyword(s):  
Calcium Imaging ◽  
Place Cells ◽  
Freely Moving Rats ◽  
Calcium Indicators ◽  
Large Populations ◽  
Freely Moving ◽  
Cell Changes ◽  
Long Timescales ◽  
In Vivo Calcium Imaging

Calcium imaging using GCaMP calcium indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for many tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes and organization over time during these tasks. To our knowledge, all reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice, and, due to their larger size, rats are able to support larger implants, thereby enabling the recording of a greater number of cells. In this paper, we present the first in vivo calcium imaging from CA1 hippocampus in freely moving rats. Using GCaMP7c and the UCLA Miniscope, we demonstrate that hundreds of cells (mean 240+-90 cells per session, maximum 428 cells) can reliably be visualized and held across weeks, and that calcium events in these cells are correlated with periods of movement. We additionally show proof of method by showing that an extremely high percent of place cells (82.3%+-8.1%, far surpassing the percent seen during mouse calcium imaging) can be recorded on a navigational task, and that these place cells enable accurately decoding of animal position. Finally, we show that calcium imaging is rats is not prone to photobleaching during hour-long recordings and that cells can be reliably recorded for an hour or more per session. A detailed protocol for this technique, including notes on the numerous parameter changes needed to use Ca2+ in rats, is included in the Materials and Methods section, and implications of these advancements are discussed.

scholarly journals On the correspondence of electrical and optical physiology in in vivo population-scale two-photon calcium imaging

2019 ◽  
Author(s):  
Peter Ledochowitsch ◽  
Lawrence Huang ◽  
Ulf Knoblich ◽  
Michael Oliver ◽  
Jerome Lecoq ◽  
...  
Keyword(s):  
Calcium Imaging ◽  
Biophysical Model ◽  
Data Set ◽  
Two Photon ◽  
Simultaneous Imaging ◽  
Fluorescence Measurements ◽  
Large Populations ◽  
Intractable Problem ◽  
Mouse Lines

AbstractMultiphoton calcium imaging is commonly used to monitor the spiking of large populations of neurons. Recovering action potentials from fluorescence necessitates calibration experiments, often with simultaneous imaging and cell-attached recording. Here we performed calibration for imaging conditions matching those of the Allen Brain Observatory. We developed a novel crowd-sourced, algorithmic approach to quality control. Our final data set was 50 recordings from 35 neurons in 3 mouse lines. Our calibration indicated that 3 or more spikes were required to produce consistent changes in fluorescence. Moreover, neither a simple linear model nor a more complex biophysical model accurately predicted fluorescence for small numbers of spikes (1-3). We observed increases in fluorescence corresponding to prolonged depolarizations, particularly in Emx1-IRES-Cre mouse line crosses. Our results indicate that deriving spike times from fluorescence measurements may be an intractable problem in some mouse lines.

scholarly journals Automated cellular structure extraction in biological images with applications to calcium imaging data

2018 ◽  
Author(s):  
Gal Mishne ◽  
Ronald R. Coifman ◽  
Maria Lavzin ◽  
Jackie Schiller
Keyword(s):  
Calcium Imaging ◽  
Image Plane ◽  
Cellular Level ◽  
Full Potential ◽  
Imaging Data ◽  
Image Domain ◽  
Biological Images ◽  
Large Populations ◽  
In Vivo Calcium Imaging

AbstractRecent advances in experimental methods in neuroscience enable measuring in-vivo activity of large populations of neurons at cellular level resolution. To leverage the full potential of these complex datasets and analyze the dynamics of individual neurons, it is essential to extract high-resolution regions of interest, while addressing demixing of overlapping spatial components and denoising of the temporal signal of each neuron. In this paper, we propose a data-driven solution to these challenges, by representing the spatiotemporal volume as a graph in the image plane. Based on the spectral embedding of this graph calculated across trials, we propose a new clustering method, Local Selective Spectral Clustering, capable of handling overlapping clusters and disregarding clutter. We also present a new nonlinear mapping which recovers the structural map of the neurons and dendrites, and global video denoising. We demonstrate our approach on in-vivo calcium imaging of neurons and apical dendrites, automatically extracting complex structures in the image domain, and denoising and demixing their time-traces.

scholarly journals In vivo wide-field calcium imaging of mouse thalamocortical synapses with an 8 K ultra-high-definition camera

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Eriko Yoshida ◽  
Shin-Ichiro Terada ◽  
Yasuyo H. Tanaka ◽  
Kenta Kobayashi ◽  
Masamichi Ohkura ◽  
...  
Keyword(s):  
Calcium Imaging ◽  
Wide Field ◽  
High Definition

scholarly journals Promoter Strength Properties of the Complete Sigma E Regulon of Escherichia coli and Salmonella enterica

2009 ◽  
Vol 191 (23) ◽  
pp. 7279-7287 ◽  
Author(s):  
Vivek K. Mutalik ◽  
Gen Nonaka ◽  
Sarah E. Ades ◽  
Virgil A. Rhodius ◽  
Carol A. Gross
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase Ii ◽  
Outer Membrane ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Strength Properties ◽  
Promoter Strength ◽  
Α Subunit ◽  
Close Relatives

ABSTRACT The σE-directed envelope stress response maintains outer membrane homeostasis and is an important virulence determinant upon host infection in Escherichia coli and related bacteria. σE is activated by at least two distinct mechanisms: accumulation of outer membrane porin precursors and an increase in the alarmone ppGpp upon transition to stationary phase. Expression of the σE regulon is driven from a suite of approximately 60 σE-dependent promoters. Using green fluorescent protein fusions to each of these promoters, we dissected promoter contributions to the output of the regulon under a variety of in vivo conditions. We found that the σE promoters exhibit a large dynamic range, with a few strong and many weak promoters. Interestingly, the strongest promoters control either transcriptional regulators or functions related to porin homeostasis, the very functions conserved among E. coli and its close relatives. We found that (i) the strength of most promoters is significantly affected by the presence of the upstream (−35 to −65) region of the promoter, which encompasses the UP element, a binding site for the C-terminal domain of the α-subunit of RNA polymerase; (ii) ppGpp generally activates σE promoters, and (iii) σE promoters are responsive to changing σE holoenzyme levels under physiological conditions, reinforcing the idea that the σE regulon is extremely dynamic, enabling cellular adaptation to a constantly changing environment.

In Vivo Calcium Imaging of Neural Network Function

Physiology ◽  
2007 ◽  
Vol 22 (6) ◽  
pp. 358-365 ◽  
Author(s):  
Werner Göbel ◽  
Fritjof Helmchen
Keyword(s):  
Calcium Imaging ◽  
Activity Patterns ◽  
Network Activity ◽  
Network Function ◽  
Two Photon ◽  
Calcium Indicators ◽  
Recent Developments ◽  
In Vivo Calcium Imaging ◽  
Function Network

Spatiotemporal activity patterns in local neural networks are fundamental to brain function. Network activity can now be measured in vivo using two-photon imaging of cell populations that are labeled with fluorescent calcium indicators. In this review, we discuss basic aspects of in vivo calcium imaging and highlight recent developments that will help to uncover operating principles of neural circuits.

scholarly journals Calcium-Permeable Presynaptic Kainate Receptors Involved in Excitatory Short-Term Facilitation Onto Somatostatin Interneurons During Natural Stimulus Patterns

2009 ◽  
Vol 101 (2) ◽  
pp. 1043-1055 ◽  
Author(s):  
H. Y. Sun ◽  
A. F. Bartley ◽  
L. E. Dobrunz
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Inhibitory Neuron ◽  
Kainate Receptors ◽  
Short Term ◽  
Natural Stimulus ◽  
Schaffer Collateral ◽  
Initial Release ◽  
Green Fluorescent

Schaffer collateral synapses in hippocampus show target-cell specific short-term plasticity. Using GFP-expressing Inhibitory Neuron (GIN) transgenic mice that express enhanced green fluorescent protein (EGFP) in a subset of somatostatin-containing interneurons (SOM interneurons), we previously showed that Schaffer collateral synapses onto SOM interneurons in stratum (S.) radiatum have unusually large (up to 6-fold) paired-pulse facilitation. This results from a low initial release probability and the enhancement of facilitation by synaptic activation of presynaptic kainate receptors. Here we further investigate the properties of these kainate receptors and examine their effects on short-term facilitation during physiologically derived stimulation patterns, using excitatory postsynaptic currents recorded in S. radiatum interneurons during Schaffer collateral stimulation in acute slices from juvenile GIN mice. We find that GluR5 and GluR6 antagonists decrease short-term facilitation at Schaffer collateral synapses onto SOM interneurons with no additive effects, suggesting that the presynaptic kainate receptors are heteromers containing both GluR5 and GluR6 subunits. The calcium-permeable receptor antagonist 1-napthyl acetyl spermine (NASPM) both mimics and occludes the effect of the kainate receptor antagonists, indicating that the presynaptic kainate receptors are calcium permeable. Furthermore, Schaffer collateral synapses onto SOM interneurons show up to 11-fold short-term facilitation during physiologically derived stimulus patterns, in contrast to other interneurons that have less than 1.5-fold facilitation. Blocking the kainate receptors reduces facilitation in SOM interneurons by ∼50% during the physiologically derived patterns and reduces the dynamic range. Activation of calcium-permeable kainate receptors containing GluR5/GluR6 causes a dramatic increase in short-term facilitation during physiologically derived stimulus patterns, a mechanism likely to be important in regulating the strength of Schaffer collateral synapses onto SOM interneurons in vivo.

scholarly journals Reconstitution of Herpes Simplex Virus Microtubule-Dependent Trafficking In Vitro

2006 ◽  
Vol 80 (9) ◽  
pp. 4264-4275 ◽  
Author(s):  
Grace E. Lee ◽  
John W. Murray ◽  
Allan W. Wolkoff ◽  
Duncan W. Wilson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fluorescent Protein ◽  
Neuronal Cell ◽  
Time Lapse ◽  
Neuronal Cell Body ◽  
Anterograde Transport ◽  
Simplex Virus

ABSTRACT Microtubule-mediated anterograde transport of herpes simplex virus (HSV) from the neuronal cell body to the axon terminal is crucial for the spread and transmission of the virus. It is therefore of central importance to identify the cellular and viral factors responsible for this trafficking event. In previous studies, we isolated HSV-containing cytoplasmic organelles from infected cells and showed that they represent the first and only destination for HSV capsids after they emerge from the nucleus. In the present study, we tested whether these cytoplasmic compartments were capable of microtubule-dependent traffic. Organelles containing green fluorescent protein-labeled HSV capsids were isolated and found to be able to bind rhodamine-labeled microtubules polymerized in vitro. Following the addition of ATP, the HSV-associated organelles trafficked along the microtubules, as visualized by time lapse microscopy in an imaging microchamber. The velocity and processivity of trafficking resembled those seen for neurotropic herpesvirus traffic in living axons. The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Immunocytochemical studies revealed that the majority of HSV-containing organelles attached to the microtubules contained the trans-Golgi network marker TGN46. This simple, minimal reconstitution of microtubule-mediated anterograde traffic should facilitate and complement molecular analysis of HSV egress in vivo.

scholarly journals Wide-field imaging of cortical neuronal activity with red-shifted functional indicators during motor task execution

2018 ◽  
Author(s):  
Elena Montagni ◽  
Francesco Resta ◽  
Emilia Conti ◽  
Alessandro Scaglione ◽  
Maria Pasquini ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Cortical Neurons ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Motor Task ◽  
Free Calcium ◽  
Wide Field ◽  
Task Execution ◽  
Additional Advantage

AbstractIntracellular concentration of free calcium ions in neuronal populations can be longitudinally evaluated by using fluorescent protein indicators, called genetically encoded calcium indicators (GECIs). GECIs with long emission wavelengths are particularly attractive for deep tissue microscopy in vivo, and have the additional advantage of avoiding spectral overlap with commonly used neuronal actuators like Channelrhodopsin.Here we investigated the performances of selected red-shifted GECIs through an ex vivo characterization and in vivo imaging of cortical mouse activity during motor task execution. Cortical neurons were infected with adeno-associated virus (AAV) expressing one of the red GECI variants (jRCaMP1a, jRCaMP1b, jRGECO1a, jRGECO1b). First we characterized the transfection in terms of extension and intensity using wide-field fluorescence microscopy. Next, we used RCaMP1a to analyse the cortical neuronal activity during motor behaviour. To that end, wide-field fluorescent microscopy and a robotic device for motor control were combined for simultaneous recording of cortical neuronal-activity, force applied and forelimb position during task execution.Our results show that jRCaMP1a has sufficient sensitivity to monitor in vivo neuronal activity over multiple functional areas, and can be successfully used to perform longitudinal imaging in awake mice.

scholarly journals Evolving small-molecule biosensors with improved performance and reprogrammed ligand specificity using OrthoRep

2021 ◽  
Author(s):  
Alex A Javanpour ◽  
Chang C Liu
Keyword(s):  
Small Molecule ◽  
High Throughput Screening ◽  
High Performance ◽  
Dynamic Range ◽  
Yeast Cells ◽  
Pathway Engineering ◽  
Muconic Acid ◽  
Metabolic Pathway Engineering ◽  
Operational Range

Genetically-encoded biosensors are valuable for the optimization of small-molecule biosynthesis pathways, because they transduce the production of small-molecule ligands into a readout compatible with high-throughput screening or selection in vivo. However, engineering biosensors with appropriate response functions and ligand specificities remains challenging. Here, we show that the continuous hypermutation system, OrthoRep, can be effectively applied to evolve biosensors with high dynamic range, reprogrammed activity towards desired non-cognate ligands, and proper operational range for coupling to biosynthetic pathways. In particular, we encoded the allosteric transcriptional factor, BenM, on OrthoRep such that propagation of host yeast cells resulted in BenM's rapid and continuous diversification. When these cells were subjected to cycles of culturing and sorting on BenM activity in the presence and absence of its cognate ligand, muconic acid, or the non-cognate ligand, adipic acid, we obtained multiple BenM variants that respond to their corresponding ligands. These biosensors outperform previously-engineered BenM-based biosensors by achieving substantially greater dynamic range (up to ~180-fold-induction) and broadened operational range. Expression of select BenM variants in the presence of a muconic acid biosynthetic pathway demonstrated sensitive biosensor activation without saturating response, which should enable pathway and host engineering for higher production of muconic and adipic acids. Given the streamlined manner in which high-performance and versatile biosensors were evolved using OrthoRep, this study provides a template for generating custom biosensors for metabolic pathway engineering and other biotechnology goals.

Sign in / Sign up

Export Citation Format

Share Document